1. Field of the Invention
The presently claimed invention relates to the use of polymerizable lipid amphiphiles in the immobilization of proteins and enzymes onto lipid polymers by non-covalent hydrogen bonding (i.e., electrostatic forces). More particularly, the presently claimed invention relates to the synthesis of polymerizable lipids and the formation of lipid vesicles (e.g. liposomes) used to immobilize enzymes and proteins via a metal ion bridge. The metal ion is chelated to functionalities on both the lipid vesicles and to amino acid residues of proteins or enzymes.
2. Description of the Related Art
Polymerizable phospholipids have been used in the stabilization of molecular assemblies and in the development of strategies to expand the usefulness of the lipid assemblies. See Singh, A., Schnur, J. M., POLYMERIZABLE PHOSPHOLIPIDS IN PHOSPHOLIPID HANDBOOK, Cevc, C., Ed., Marcel Dekker Inc., N.Y. pp. 233-287 (1993). See Ringsdorf, H., Schlarb, B., Venzmer, J., 27 ANGEW. CHEM. INT. ED. ENGL. pp. 113 (1988). These strategies include alteration of surface properties of polymer films and construction of a polymerized lipid-membrane surface equipped with reactive sites suitable for chemical interactions, See Regen, S. L., Kirzenstejn, P., Singh, A., 16 MACROMOLECULES pp. 335 (1983) incorporated herein by reference in its entirety and for all purposes. See Markowitz, M. A., Schnur, J. M. and Singh, A., 62 CHEM. PHYS. LIPIDS pp. 193 (1992). See Markowitz, M. A., Baral, S., Brandow, S., and Singh, A., 224 THIN SOLID FILMS pp. 242 (1983). U.S. Pat. No. 5,258,499 (Konigsberg et al.) discloses the use of a liposome. In the Konigsberg et al. patent, the invention includes a composition of matter comprising a liposome which is covalently hound to a ligand, for example, a protein or an enzyme. (Column 2, lines 40-47). The ligands are covalently attached to the liposome surface by coupling agents which are covalently bonded to the ligand. (Column 4, lines 1-5). U.S. Pat. No. 4,913,902 (Kilpatrick et al.) discloses the use of liposomes as well. Liposomes are used to hind various ligands that exist in solution. Thereafter, the liposome-ligand(s) are extracted from solution by filtration. In this manner, the various ligands are extracted from solution by the use of the claimed liposomes of Kilpatrick et al. At column 3, lines 66-68 and column 4, lines 1-5, it is stated that
These ligands may be covalently bound to phospholipids used to form liposomes by conventional techniques, either by attracting the ligand to preformed liposomes or by binding the ligand to a phospholipid and incorporating the resulting amphiphilic molecules into liposomes during formation thereof. PA1 Ligands which will bind to any of a variety of target molecules can be bound to liposomes to practice the present invention. Exemplary of such ligands, and the target molecules bound thereby, are the following: . . . Chelating Agents and Metal Ions . . .
At column 3, lines 9-14 and lines 34-36 it is further stated that
The Kilpatrick et al. patent discloses the use of a ligand which may be covalently bound to the liposome to remove a target molecule. The covalently bound ligand, for example, a chelating agent, may be used to remove a target molecule, for example, a metal ion.
Various other papers disclose the covalent binding of ligands to liposomes or to other materials, such as glass microbeads or polymers. See Wasserman et al., High-Yield Method for Immobilization of Enzymes, 22 BIOTECHNOLOGY AND BIOENGINEERING pp. 271-287 (1980); Epton et al., A Study of Carbonic Anhydrase Covalently Bound to a Poly (acryloylmorpoline) Network in Aqueous/Organic Solvents, 5(1) BIOCHEMICAL SOCIETY TRANSACTIONS pp. 274-276 (1977); Crumbliss et al., Preparation and Activity of Carbonic Anhydrase Immobilized on Porous Silica Beads and Graphite Rods, 31 BIOTECHNOLOGY AND BIOENGINEERING pp. 796-801 (1988); Cantenys et al., Covalent Attachment of Insulin to the Outer Surface of Liposomes, 117 (2) BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, pp. 399-405 (Dec. 16, 1983); Martin et al., Irreversible Coupling of lmmunoglobulin Fragments to Preformed Vesicles, 257(1) THE JOURNAL OF BIOLOGICAL CHEMISTRY pp. 286-288 (Jan. 10, 1982); and O'Daly et al., Activity of Carbonic Anhydrase Immobilized on Porous Silica Beads in Organic Media, 12 BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, pp. 11-19 (1990). A disadvantage of covalently immobilizing enzymes or proteins on liposomes is that the activity of the enzymes may be altered or substantially decreased. Therefore, it is desirable to provide a means for transporting enzymes and proteins that are immobilized by non-covalent binding.
In U.S. Pat. No. 4,867,917 (Schnur and Singh), incorporated herein in its entirety and for all purposes, a method for synthesizing diynoic acid having the formula: EQU CH.sub.3 (CH.sub.2).sub.n --C.tbd.C--C.tbd.C--(CH.sub.2).sub.m COOH(1)
was disclosed wherein n and m are integers from 5-11 and 7-16, respectively. The basic heterocoupling reaction achieved for the formation the above-identified diynoic acids is between: EQU HC.tbd.C--(CH.sub.2).sub.m --COOH and CH.sub.3 --(CH.sub.2).sub.n --C.tbd.C--X
wherein X is a selected halogen, preferably iodo, bromo or chloro and where m is 5,6,7,8,9,10, or 11 and where n is 7,8,9,10,11,12,13,14,15, or 16. See column 2, lines 45-70 and the remainder of the Schnur and Singh U.S. Pat. No. 4,867,917 for the synthetic scheme, incorporated herein by reference. It should be noted that the same synthetic scheme outlined in U.S. Pat. No. 4,867,917 for forming the diynoic acids wherein n is between 7-16 and m is between 5-11 can also be successfully used for forming diynoic acids wherein n is between 1-27 (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) and wherein m is between 2-17 (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17).
These diynoic acids were useful in forming certain alkadiynoyl-sn-glycero-3-phospholipids. The phospholipids derived from the diynoic acids allow polymerization thereof to form certain microstructures which are peculiarly stable in harsh physical and chemical environments. In particular, the microstructures may be lipid bilayers, tubules, liposomes of various diameters and other microstructures. U.S. Pat. No. 4,867,917 (Schnur and Singh) is incorporated herein by reference in its entirety and for all purposes. The microstructure of phospholipids having the formula: ##STR2## where n equals 2 or 3 or 4 are discussed by Markowitz, Schnur and Singh. See Markowitz, M., Schnur, J., and Singh, A., The influence of the polar headgroups of acidic diacetylenic phospholipids on tubule formation, microstructure morphology and Langmuir film behavior, 62 CHEMISTRY AND PHYSICS OF LIPIDS, pp. 193-204 (1992), incorporated herein by reference in its entirety and for all purposes. See also Markowitz, M., Baral, S., Brandow, S., and Singh, A., Palladium ion assisted formation and metallization of lipid tubules, 224 THIN SOLID FILMS pp. 242-247 (1993), incorporated herein by reference in its entirety and for all purposes. See also Singh, A., and Schnur, J. M., CHAPTER 7-POLYMERIZABLE PHOSPHOLIPIDS in PHOSPHOLIPIDS HANDBOOK, Gregor Cevc (Editor), Marcell Dekker, Inc., New York, pp. 233-291 (1993), incorporated herein by reference in its entirety and for all purposes. It is known that covalent enzyme immobilization on surfaces usually results in substantial loss of enzyme activity. See Wulf G,, POLYMERIC REAGENTS AND CATALYSIS, Ford, W. (Editor), ACS Symposium Series 308, American Chemical Society, pp. 186-230, Washington, D.C. (1986). See also Sinha, P. et al., 28A IND. J. CHEM. PP. 33515 (1989). Thus, there is a need for a means to immobilize proteins and/or enzymes by non-covalent binding upon a microstructure such as a liposome or a lipid bilayer wherein the problem of a substantial loss of, for example, enzyme activity associated with covalent binding, is avoided. To date, phosphatidylcholine derivatives for forming polymerized liposomes or bilayers comprising polymerizable metal chelating lipids interspersed within the liposomes or lipid bilayers formed have not been made. There is a need for forming polymerizable metal chelating lipids that can be incorporated into a microstructure, for example, a liposome or a lipid bilayer. There is a need for forming microstructures, for example, liposomes or bilayers wherein polymerizable metal chelating lipids are incorporated into the liposome or the bilayer wherein the polymerizable metal chelating lipid can chelate with metal ions which metal ions can further chelate with proteins or enzymes resulting in non-covalent immobilization of, for example, a protein or an enzyme onto the microstructure. There is a need for forming lipids capable of forming microstructures such as liposomes or bilayers, capable of non-covalently immobilizing proteins and/or enzymes and capable of being polymerized so that the microstructures formed (e.g. liposome or bilayers) do not break apart upon, for example, mild sonication or under working conditions, for example, of pH between about 2 to 11 and temperature of between about 0.degree. C. to about 80.degree. C. See R. L Juliano, S. L Regen, M. Singh, M. J. Hsu and A. Singh, Stability properties of photopolymerized liposomes, 1 BIOTECHNOLOGY pp. 882-885 (1983). There is a need for forming polymerized liposomes of variable diameter that are formed from polymerizable non-metal chelating lipids interspersed with polymerizable metal chelating lipids having moieties capable of chelating with metal ions, which metal ions are further capable of chelating with proteins and/or enzymes in order to non-covalently immobilize the proteins and/or enzymes.